The NCI, CCR, LGI Flow Cytometry Core Facility has provided service to 83 Principle Investigators and 236 users from 31 NCI labs or branches this year. The major focus of NCI investigators using the Flow Cytometry Core Facility is to identify and study cancer cells. A number of NCI labs are using flow cytometry and fluorescence-activated cell sorting to identify and sort the cancer stem cell by membrane antigen expression using monoclonal antibodies or with a functional assay involving active membrane substrate transport. Investigators in CCBB, MBTL, LEC, LHC, and LCBG are studying cancer stem cells from breast, ovarian, hepatic, thyroid, pancreatic and lung carcinomas. The BD LSRII, BD LSRFortessa flow cytometer cell analyzers, the special order BD FACSAria, and the BC MoFlo Astrios cell sorters are frequently used for these assays. Transfection of cells with genes expressing fluorescent reporters is a technique used by the majority of labs using the facility. The Core cytometers and cell sorters have been equipped with specific lasers to allow detection and sorting of cells labeled with green, yellow, blue or red fluorescent proteins or with combinations of these fluorescent reporters. Sorted transfected cells are used to prepare protein, DNA, and RNA that can be used in Western blotting and microarrays. Sorted cells are also used to determine effects of siRNA, to look at signaling proteins, or may be further passaged to create stable cell lines. Fluorescent reporter proteins may also be linked to luciferase. Tumor cell lines have been sorted based on their expression of green or red fluorescent protein to establish cell lines with high levels of expression. These cell lines have then been used to establish tumors in mice and to image metastasis. Flow cytometry is also frequently used for looking at cell growth (cell cycle analysis) and mechanisms of cell death (apoptosis) to examine actions of cancer drugs. Last year the Flow Cytometry Core Facility implemented the new policy for cell sorting. According to the NIH Policy for Biosafety of Cell Sorters (July 28, 2012), sorting of human cell lines, human cells and 2nd or 3rd generation Lentivirus in human cells require the sort to be performed at a BSL-2 with Enhanced Precautions biosafety level. The Beckman Coulter MoFlo Astrios cell sorter was upgraded with a custom designed SterilGARD Class II Biosafety Cabinet with an onboard HEPA filtration system that is uniquely configured to fit on the instrument bench to provide safe, effective aerosol evacuation. This instrument upgrade enables the flow cytometry staff to perform sorts in compliance with the new guidelines. A BD FACSAria Fusion cell sorter has been ordered for the purpose of providing this same level of safety when sorting samples of this type. A Class II type A2 Biosafety Cabinet is integrated into the instrument to ensure that aerosols are always contained and evacuated into the HEPA filters. Both cell sorters will be housed in the newly renovated space that is designated for sorts requiring BSL-2 with Enhanced Precautions. In addition to providing superior biosafety protection the MoFlo Astrios and FACSAria Fusion each have 5 lasers and 18 detectors capable of sorting cells stained with various combinations of fluorochromes. Cells can be sorted as single cells into 96-well plates or bulk sorting into tubes. When additional sorts require BSL-2 with Enhanced Precautions, Core staff restrict entry to the facility and use powered air purifying respirators (PAPR's) to preform sorts of this kind. Beckman Coulter upgraded the MoFlo Astrios to enable the detection of nano particles. Several investigators are interested in analyzing and sorting exosomes. This is an application that will be developed over the next year. Appointments to use the analyzers are scheduled with shared Microsoft Outlook calendars. The cell sorters are scheduled after the NCI, CCR, LGI Flow Cytometry Core Facility Biosafety Questionnaire and Sort Request form has been submitted and reviewed by the Core Manager. The core website, located at https://labshare.nih.gov/nci/ccr-facs is currently under construction. It will soon contain pertinent information about the services the core offers as well as provide an easy scheduling system.